Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used negative staining and cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. We have examined the structure of actin-bound myosin V mutants in which the neck length was altered. These studies demonstrate that the neck of myosin V has evolved to allow it to take 36 nm strides along actin which matches the helical repeat. Atomic force microscopy was used to study the extensibility of the tails of myosin V from Drosophila and mouse. In both cases, the force-extension curve of these myosin tails differed dramatically from that of rabbit skeletal muscle myosin in that less force is needed to bring about extension. There were also differences in the behavior of the tail from the processive mouse myosin V and the nonprocessive Drosophila myosin V. Modeling studies have shown that an extensible myosin V tail would be very useful for processive movement in a viscous environment. We have mutated the switch-1 region of myosin V (S217A) and found that this mutant alters the duty ratio and effects processivity of the motor. Switch-2 mutants affect the speed of translocation and the ADP release rate. Optical trapping studies show that under certain levels of strain, the powerstroke of myosin V attached to actin can be reversed which may aid in maintaining myosin attachment during stall.